The FASEB Journal express article10.1096/fj.03-0376fje. Published online October 2, 2003.

Expression of the small heat-shock protein Hsp-16-2 in

Caenorhabditis elegans is suppressed by Ginkgo biloba

extract EGb 761

Amy Strayer,* Zhixin Wu,* Yves Christen,� Christopher D. Link,

� and Yuan Luo*

*Department of Biological Sciences, The University of Southern Mississippi, Hattiesburg, MS; �Beaufour Ipsen, Paris, France;

�Institute for Behavioral Genetics, University of Colorado,

Boulder, CO

Corresponding author: Yuan Luo, Department of Biological Sciences, 2609 West 4th Street, The

University of Southern Mississippi, Hattiesburg, MS 39406-5018. E-mail: yuan.luo@usm.edu

ABSTRACT

EGb 761, a standardized extract of Ginkgo biloba leaves, has been shown to have antioxidative

properties. We have previously demonstrated that EGb 761 increases stress resistance and mean

life span in the model organism Caenorhabditis elegans. In this study, the molecular mechanism

of EGb 761 on alleviating effects of oxidative stress is further investigated using transgenic C.

elegans expressing a jellyfish green fluorescent protein (GFP)-tagged inducible small heat-shock

protein gene (hsp-16-2). The expression of hsp-16-2 induced by the pro-oxidant juglone and by

heat shock was significantly suppressed by 86% and 33%, respectively, in the transgenic

nematode fed with EGb 761. These effects of EGb 761 correlate with its ability to increase mean

survival rate of the nematode in response to acute oxidative and thermal stresses, as well as to

attenuate the basal levels of hydrogen peroxide in the organism. Thus, we interpret the

suppression of hsp-16-2/GFP expression as an indication that EGb 761 decreases cellular stress

resulting from exogenous treatments, therefore leading to a decreased transcriptional induction of

the reporter transgene. These results support the hypothesis that EGb 761 augments the natural

antistress system of C. elegans, thus increasing stress resistance and life span.

Key words: GFP reporter � ROS � oxidative stress

ll organisms have developed the ability to respond to environmental threats by the

synthesis of highly conserved stress response proteins. A universal response to elevated

temperature and other forms of stress is the induction of heat-shock proteins (HSPs).

These include HSP-100, HSP-90, HSP-70, HSP-60, HSP-40, HSP-30, and the small HSPs

(sHSPs) (1). The sHSPs belong to a family of low molecular weight polypeptides (12�43 kD)

that have been highly conserved from yeast through humans and show sequence as well as

functional similarities to the lens α B crystallins (2). Under physiological conditions, sHSPs are

among the most highly inducible HSPs during thermal stress or oxidative stress. Their expression

correlates specifically with the presence of stressors (3) that induce protein damage (4). In the

nematode C. elegans, 16 sHSPs have been identified. The major sHSPs of the 16 kD species

(hsp-16-1, hsp-16-2, hsp-16-41, and hsp-16-48) are only expressed under stress conditions (5). A

mutagenesis study of the hsp-16-2 promoter has demonstrated that HSF, and possibly other

A

transcription factors, control hsp-16-2 induction in response to heat shock (6). To follow sHSP

regulation in vivo, we constructed transgenic reporter strains (e.g., CL2070) in which the

transcription of a jellyfish green fluorescent protein (GFP) is driven by the promoter of the hsp-

16-2 gene (7). Expression of the GFP in the CL2070 reporter strain parallels endogenous

expression of hsp-16-2 protein, and thus the response of this strain to various stressors can be

quantitatively assayed in living animals.

The standard Ginkgo biloba leaf extract EGb 761 is a popular dietary supplement taken by the

general population to enhance mental focus and by the elderly to delay the onset of age-related

loss of cognitive function. During the past decade, in vivo and in vitro experiments in

mammalian systems and clinical studies in humans demonstrated that EGb 761 exhibits a range

of biochemical and pharmacological effects that include cognition enhancement and stress

alleviation (8). In human studies, available data have confirmed the clinical efficacy of EGb 761

in primary degenerative dementia of Alzheimer�s type (9�11). Some data support the view that

the extract enhances learning and longevity in rats (12) and has neuromodulatory and

neuroprotective properties in several species (13�16). However, the evidence of an effect on

memory in healthy humans is still inconclusive (17); some studies found an effect (18), and

others did not (19). Currently there are no data about longevity in humans.

We have previously reported that EGb 761 protects cultured neuronal cells from stress-induced

cell death (20, 21), increases stress resistance and mean life span in model C. elegans (22), and

serves as a stress buffer in experimental mice (23). Because sHSPs are a family of molecular

chaperones that are expressed only under stress conditions, we asked whether EGb 761 affects

the expression of these proteins in C. elegans. Therefore, we used the CL2070 transgenic GFP

reporter strain to visualize sHSP expression in living animals in real time. Here, we demonstrate

that the stressor-induced expression of hsp-16-2 is suppressed in C. elegans fed with EGb 761,

suggesting a modulatory role of the extract in the function of a stress-response gene.

Furthermore, with the combination of the survival assay and the assay for levels of oxidative free

radicals in C. elegans, our results indicate that the effect of EGb761 on the modulation of hsp-

16-2 expression is beyond its known function as a scavenger for oxidative free radicals.

MATERIALS AND METHODS

Gingko biloba

The standardized leaf extract EGb 761, consisting of two major active constituents (8), flavonols

(24%) and terpene lactones (6%), was provided by Schwabe Pharmaceuticals (Karlsruhe,

Germany). The isoflavone constituent was a gift from Beaufour IPSEN (Paris, France). The

terpene lactones, including GA, GB, GC, GJ, and BB, were obtained from Dr. Ikhlas Khan of the

National Center for Natural Products Research (University, MS) (24). Stock solutions of EGb

761 (1000×) were made in 100% ethanol. The final concentration of ethanol did not exceed

0.01% in the food (Escherichia coli strain OP50). Juglone, 5-hydroxy-1,4-napthoquinone, and L-

ascorbate, were obtained from Sigma Pharmaceuticals (St. Louis, MO).

Caenorhabditis elegans strains, maintenance, and treatment

The wild-type N2 strain was obtained from the Caenorhabditis Genetics Center, University of

Minnesota (Minneapolis, MN). The transgenic strain hsp-16-2/GFP (CL2070) was generated and

characterized by Dr. C. Link as described previously (7). CL2070 contains a jellyfish GFP

reporter transgene that is under the control of the promoter for the sHSP gene hsp-16-2. The HSP

hsp-16-2 is expressed by either heat shock (35°C for 2 h) or by exposure to juglone (40 µM for

24 h), a quinone known to induce superoxide radicals in C. elegans. All nematodes were

cultivated on nematode growth medium (NGM) agar on 60 mm Petri plates and maintained at

20°C in a temperature-controlled incubator. E. coli (OP50) was the food source and was added to

the surface of NGM plates at 100 µl. To obtain age-synchronized nematodes, we left the self-

fertilizing hermaphrodites to lay eggs (for 4�8 h) on their third day of life and then removed

them to set the eggs in synchrony. For the pretreatment protocol, the nematodes were fed EGb

761 on the day after hatching (except life span assay) for 48 h, and the stressors were applied on

their third day of life when they passed into the adult stage. Stock solutions of juglone were

made as 1 mg/1 ml in 100% ethanol. Juglone was mixed freshly with NGM to reach the desired

final concentration and dried to administer the stressor exogenously through the skin.

Fluorescence microscopy and quantitation of hsp-16-2 expression

Using the CL2070 strain, we used thermal or oxidative stress to induce hsp-16-2 gene

expression. For heat shock, the hsp-16-2/GFP worms, synchronized and maintained as stated

above, were exposed to 35°C for 2�4 h. Worms were then allowed to recover in their normal

environment at 20°C for 12 h before pictures were taken. The oxidative stress involves exposing

the worms to 40 µM juglone for 24 h. After both inductions, the expression of hsp-16-2 was

measured by directly observing the fluorescence of the reporter GFP. Epifluorescence images

were acquired at the same exposure parameter using the 40× objective of a microscope (BX 60,

Olympus, Tokyo, Japan) equipped with a digital camera (Micropublisher 5.0, QIMAGING,

Burnaby, BC, Canada). For quantifying a population of GFP reporter animals, each 40× image

was analyzed using Image-ProPlus 4.51 software (MediaCybernetics, Silver Spring, MD).

Survival assays

Thermotolerance was measured using a heat stressor. Worms (3 dishes of 30 worms each) were

treated with 100 µg/ml EGb 761 for 48 h and were then exposed to 35°C for 2�4 h. The number

of survivors was counted every hour until all were dead. Oxidative stress was induced by an

acute, lethal concentration of juglone at 160 µM. Worms were first treated with 100 µg/ml EGb

761 on the day after hatching and then were transferred to fresh dishes after 2 days of treatment.

Juglone was added to liquefied NGM at 65°C and then pipetted to 35 mm Petri plates. After the

plates had solidified, OP50 was added at 70 µl and the plates were dried in a fume hood. Worms

were transferred within an hour of preparing the dishes and were counted every 30 min for

survival until all were dead. They were scored dead if they did not respond to a touch stimulus.

Analysis of oxidative free radicals

Intracellular hydrogen peroxide (H2O2)-related reactive oxygen species (ROS) were measured in

C. elegans using 2,7-dichlorofluorescein diacetate (DCF-DA; Molecular Probes). Nonfluorescent

DCF-DA is a cell-permeable dye that is readily converted to 2,7-dichlorofluoroscein (DCF) by

interacting predominantly with hydrogen peroxide (25). Age-synchronized C. elegans treated

with or without EGb 761 on the day after hatching for 72 h were collected into 100 µl PBST

(PBS containing 0.1% Tween 20) with 30 worms from each group and 3 groups per treatment.

The worms were then subjected to timed homogenization (Pellet Pestle Motor, MG Scientific)

and sonication (Branson Sonifier 250, VWR Scientific) to break up the outer cuticle. Samples

were vortexed, transferred into 96-well plates, and incubated with 50 µM DCF-DA in PBS at

37°C in a fluorescent microplate reader (Bio-Tek Instruments, Winookski, VT) for quantification

of fluorescence at excitation 485 nm and emission 640 nm. Samples were read kinetically every

10 min for 2.5 h.

Statistical analyses

Statistical comparison between treatments was done with unpaired Student�s t test using Origin

6.0 software (Microcal Software, Northampton, MA). Standard error of the mean was used in the

figures. Differences of P<0.05 were defined as statistically significant.

RESULTS

Stress-induced expression of hsp-16-2/GFP is suppressed in the transgenic C. elegans fed

with EGb 761

We have previously shown that EGb 761 increases stress resistance in the model C. elegans (22).

To determine whether this is due to EGb 761 regulating a specific stress-response gene, we used

the transgenic C. elegans (CL2070) expressing GFP as a reporter transgene for inducible hsp-16-

2 expression. Figure 1A shows the phenotype of wild-type (a) and the transgenic strain CL2070

(b), in which the hsp-16-2/GFP gene expression was induced by a rise in temperature from 20°C

to 35°C for 2 h. Upon induction by thermal stress, the GFP fluorescence is visible at the head of

the worm, including the pharynx and the anterior nerve ring in the transgenic worm (b) but not in

the wild-type controls (a).

The expression of hsp-16-2 induced by heat shock was significantly suppressed by 33% in

CL2070 worms fed with EGb761 (control, GFP mean pixel density 75 vs. EGb-treated, GFP

mean pixel density 50, n=72 worms, P<0.05, Fig. 1B). Figure 1B insets: representative hsp-16-

2/GFP expression induced by heat shock in CL2070 worms untreated (a) or treated with 100

µg/ml EGb 761 (b). Exposure of the transgenic worms to 40 µM juglone, an oxidative stressor,

for 24 h generated higher hsp-16-2 expression (Fig. 1C, inset a) than that induced by heat shock

(Fig. 1B, inset a). Shorter exposure time to juglone also induced hsp-16-2 expression but to a

lesser degree: an 11% and 30% induction were observed in worms exposed to juglone for 6 h

and 12 h, respectively (24 h exposure set as 100%, data not shown). Most importantly, the

juglone-induced expression of hsp-16-2 was remarkably attenuated by 86% in worms pretreated

with 100 µg/ml EGb 761, compared with untreated controls (EGb761, GFP mean pixel density

180 vs. control GFP mean pixel density 25, n=120 worms, P<0.001, Fig. 1C). Similar results

were obtained in worms exposed to a higher concentration of juglone (60 µM, data not shown).

It is possible that the attenuation of hsp-16-2/GFP expression by EGb 761 could be due to the

drug�s interference with reporter GFP expression per se. To exclude this possibility, we used a

chromosomally integrated transgenic line (CL1234) that constitutively expresses GFP from the

synaptobrevin snb-1 pan-neuronal promoter to serve as a control. The CL1234 worms were

treated with the same concentration of EGb 761 for the same time as CL2070 worms, and GFP

fluorescence was measured subsequently by fluorescence microscopy. Figure 1D insets show the

representative GFP expression in control (a) and EGb 761-treated (b) CL1234 worms. For

statistical analysis of GFP expression affected by EGb 761 treatment, only the anterior neurons

within the head region of the worms were outlined and analyzed. From three independent

experiments of 30 worms each, the fluorescence density was not significantly affected by the

treatment of the CL1234 worms with EGb 761 (Fig. 1D graph), strongly suggesting that the

attenuation of hsp-16-2/GFP by EGb 761 is not an artifact.

EGb 761 treatment increased survival rate in transgenic C. elegans under stress conditions

To provide further evidence that the attenuation of hsp-16-2 expression by EGb 761 treatment of

the animals is a beneficial effect, we conducted survival assays in CL2070 worms treated with or

without EGb 761 before exposure to either a thermal or an oxidative stressor. Figure 2A

demonstrates that pretreatment of the CL2070 worms with 100 µg/ml EGb 761 increased their

survival in response to the heat shock (percent survival at 15 h, control 15.0% ± 10.1 vs. EGb

48.6% ± 0.9, n=189 worms). Figure 2B shows that pretreatment of the CL2070 worms with 100

µg/ml EGb 761 increased their survival rate in response to exposure to the pro-oxidant juglone

(160 µM) (mean survival: control 4.6 h vs. EGb 5.6 h, n=179 worms). This result is consistent

with our previous observation in the wild-type C. elegans treated with EGb 761 (22) and further

indicates that the attenuation of hsp-16-2/GFP expression by EGb 761 does not cause abnormal

physiological changes, which may affect GFP reporter gene expression.

Postjuglone treatment with EGb 761 suppressed the expression of hsp-16-2

EGb 761 has been known to have dual antioxidative actions, with its flavonoid constituent able

to scavenge oxidative frees radicals and its ginkgolide constituent able to prevent the formation

of free radicals (8). To test any poststress effects of EGb 761 against oxidative damage, we

treated the worms with EGb 761 either simultaneously with or 24 h after exposure to 40 µM

juglone to induce hsp-16-2/GFP expression. Figure 3A shows that EGb 761 suppressed hsp-16-2

expression induced by concomitant juglone treatment by 66% (control, GFP mean pixel density

240 vs. EGb 761, GFP mean pixel density 80, n=2, P<0.05, total of 30 worms), and hsp-16-2

expression induced by postjuglone treatment by 50% (control, GFP mean pixel density 240 vs.

EGb 761, GFP mean pixel density 120, n=2, P<0.05, total of 35 worms). These results suggest

that EGb 761 may function downstream of juglone-generated damage, because not only does it

prevent stressor-induced hsp-16-2 gene expression, but it also suppresses gene expression after

the damage has been done.

If the suppression of juglone-induced hsp-16-2 expression by EGb 761 is due to its antioxidative

actions, then other antioxidants should exhibit the same effect. To further characterize the

involvement of antioxidative properties of EGb 761 in the modulation of hsp-16-2 expression,

we pretreated the worms with the known antioxidants L-ascorbic acid (vit C, 100 µg/ml) or the

flavonoid fractions of EGb 761 (FLV, 100 µg/ml) before exposure to 40 µM juglone. Figure 3B

demonstrates that juglone-induced hsp-16-2 expression was not significantly suppressed by

either L-ascorbic acid or the flavonoid fractions, even with the higher concentration of flavonoids

than present in the whole extract (note that the attenuation of hsp-16-2 expression by vitamin C

is close to being significant but still less than that seen with EGB 761).

EGb 761 attenuated intracellular levels of hydrogen peroxide in C. elegans

Oxidative free radicals have been postulated as a cause of aging and of some degenerative

diseases (26, 27). Profound induction of hsp-16-2 expression by juglone (Fig. 1C) suggests that

the sensing of oxidative stress triggers the induction of sHSP expression. Recently, we were able

to measure H2O2-related ROS levels in C. elegans, and we observed an age-dependent increase

in the levels of H2O2 and an increased level of H2O2 in a transgenic C. elegans model expressing

the Aβ peptide (28). To monitor the ROS levels in the transgenic C. elegans CL2070 treated with

EGb 761, we performed a DCF-DA fluorescence assay. Although we were unable to detect an

increase in ROS induced by juglone in these worms, we consistently observed (Fig. 4) that in the

CL2070 C. elegans treated with EGb 761, the basal ROS levels were significantly attenuated by

24% (control 100% vs. EGb 76%). In the CL2070 worms treated with L-ascorbic acid, H2O2-

related ROS levels were attenuated by 31% (control 100% vs. VitC 69%), and in the worms fed

with the flavonoids of EGb 761, by 30% (control 100% vs. FLV 70%). Together with the ability

of EGb 761 and the known antioxidants to suppress hsp-16-2 expression, it suggests that other

functions of EGb 761 contribute to the suppression of hsp-16-2 expression.

DISCUSSION

This study sought to delineate the antistress mechanisms of EGb 761 by using the transgenic C.

elegans strain CL2070, a model well suited for studying the regulation of a specific stress-

response gene in vivo and for examining stress response and aging (29). Our results demonstrate

that the expression of the hsp-16-2/GFP reporter gene induced by thermal and oxidative stressors

was significantly suppressed in CL2070 C. elegans (Fig. 1). This effect of EGb 761 correlated

with the ability of EGb 761 to increase the organism�s resistance to thermal and oxidative

stressors (Fig. 2) and to attenuate H2O2-related ROS levels in the whole organism (Fig. 4). We

interpret the suppression of hsp-16-2/GFP reporter transgene expression as an indication that

EGb 761 decreased cellular stress induced by exogenous stressors, leading to a decreased

transcriptional induction of the reporter transgene. Postadministration effects of EGb 761 on

suppressing hsp-16-2 expression (Fig. 3) suggest that the extract functioned not only as a

scavenger for oxidative free radicals that prevent the propagation of free radical damage, but also

as the enhancer of repair or turn-over of damaged macromolecules.

We propose that the induction of hsp-16-2 expression upon treatment with the stressors can

result from an intracellular increase in ROS or by high levels of damaged proteins (Fig. 5). It is

unlikely that the oxidative stress directly induced hsp-16-2 expression. The resulting damaged

proteins could be the actual signal. This might explain the suppression of hsp-16-2 expression by

EGb 761 long after juglone exposure (Fig. 3B), that is, EGb 761 antioxidative activity reduces

protein oxidation downstream of juglone exposure. The suppression of juglone-induced hsp-16-2

expression by EGb 761 was more profound than that of heat-shock-induced hsp-16-2 expression

(Fig. 1B and 1C), suggesting a strong antioxidative action of the extract. This effect of EGb 761

is apparently not due to affecting the expression of the reporter gene GFP per se, because a

transgenic line of C. elegans (CL1234) expressing GFP constitutively did not show a difference

in GFP expression upon treatment with EGb 761 (Fig. 1D). Furthermore, the worms treated with

EGb 761 before juglone exposure were noticeably healthier than animals treated with juglone

alone (Fig. 2B), indicating that the suppression of juglone-induced hsp-16-2 expression was not

due to the EGb 761-treated animals being �sick� and unable to produce GFP.

Our understanding of the function of sHSPs remains incomplete. A recent study indicates that

the expression of sHSPs increases life span (30). It has been suggested that induction of sHSP

expression may play a protective role in a stress situation, by binding to abnormal proteins to

disrupt or prevent their aggregation, facilitating a renaturation or repair process (31) and acting

as molecular chaperones (32). sHSPs may also prevent apoptosis (33) via modulating

intracellular glutathione level (34), phosphorylation of MAPK, and induction of the FAS

receptor-mediated pathway (35). Conceivably, strong sHSP expression could also have

deleterious longer term cellular consequences, to the point that EGb 761 attenuation of sHSP

expression is directly beneficial.

The modulation of hsp-16-2 expression by EGb 761 is beneficial, which may be important for

the adaptive function (36). If hsp-16-2 has a protective function against stress, EGb 761 might

protect against the deleterious process leading to the decreased need for hsp-16-2 expression.

The ability of EGb 761 to increase the survival rate of the C. elegans induced by a wide range of

different stimuli (Fig. 2 and ref 22) suggests an intervention at the level of general and early

mediators of cellular stress responses. EGb 761 has been postulated to act as a biological

response modifier, perhaps aiding hsp-16-2 in its functions. In an Aβ-secreting transgenic

neuroblastoma cell line, we have demonstrated that EGb 761 inhibits Aβ aggregation and Aβ-

induced apoptosis (20). One can speculate that the presence of EGb 761 reduces the cellular flux

of free radicals, leading to a concomitant decrease in damaged proteins and a reduced

requirement for HSP expression. This hypothesis was recently strengthened by reports that EGb

761 modulates HSP-70 expression in several model systems (37�39).

The results described do not address the functional interaction between EGb 761 and hsp-16-2,

but it is possible that EGb 761 functions downstream of an applied stressor. This is supported by

our observation that EGb 761 was able to suppress the expression of hsp-16-2 after 24 h of

juglone exposure (Fig. 3B). In mammalian cells, the phosphorylation of constitutively expressed

sHSPs is a first phase of stress response, while elevated sHSP expression, at a time when protein

phosphorylation is already down-regulated, comprises the second phase (31). MAP kinase has

been identified to phosphorylate sHSPs under stressful conditions (31, 40�42). EGb 761 may

thus affect events upstream from sHSP expression via inhibition of MAP kinase-mediated

signaling pathways (43). Subsequently, the extract may limit stress-induced damage by

interfering with degradative pathways, thus sparing neurons or enhancing normal cellular

protection mechanisms. The potential polyvalent activities of EGb 761 give the Ginkgo biloba

extract an advantage over conventional single-component antioxidants by offering more effective

cellular protection.

Another important result shown in this study is that the basal levels of ROS were attenuated in

the EGb 761-fed transgenic worms, compared with untreated worms (Fig. 4). This is consistent

with our recent observation that elevated levels of ROS in the AD-associated transgenic C.

elegans CL2006 are attenuated by EGb 761 administration (28). The challenges of measuring

levels of the transient oxidative free radical molecules in whole organisms are well documented

(44). Previously, we demonstrated the reliability of the assay by showing an increased level of

ROS over the life span of the whole organism C. elegans, an increased rate of ROS accumulation

in the AD-associated animals in comparison with their wild-type counterparts, and a more than

fourfold increase of ROS in an endogenous antioxidant-deficient mutant mev-1 (28). Thus, our

observation favors the hypothesis that the modulation of cellular stress response by EGb 761 is

related to its role in ROS attenuation. Note that we were unable to test H2O2-related ROS

produced by juglone with the DCF-DA protocol. There are several reasons for the inability to

measure juglone-induced ROS: 1) Juglone is known to produce the superoxide anion by

interfering with electron transport chain activity (45), whereas the DCF-DA protocol we used is

not specific for the highly transient superoxide anion but for hydrogen peroxide; 2) juglone may

interfere with tissue extraction, even at low concentrations (46), which may pose significant

problems for our assay even with, for example, longer sonication and homogenization times and

higher detergent concentrations in PBST; and 3) restricted food intake can result in lower

metabolism and, hence, fewer free radicals. During behavioral studies, it was observed that the

worms were much less mobile under juglone treatment than without (data not shown). Therefore,

the worms could not take in as much food as they would in the absence of the juglone.

Taken together, the results of this study indicate that EGb 761 modulates the expression of an

immediate stress-response gene hsp-16-2 under exogenous stress stimuli, and this effect is

beyond its known function as a scavenger for oxidative free radials. Our results suggest that

oxidative stress and the consequent oxidative damage can be successfully counteracted by the

Ginkgo biloba extract EGb 761, probably via regulation of endogenous antistress mechanisms. A

better understanding of the mechanisms of neuroprotection by EGb 761 will be important for the

basic understanding of underlying stress-response processes and for the effectiveness and

complex functions of this herbal medicine.

ACKNOWLEDGMENTS

We would like to thank Schwabe Pharmaceuticals (Karlsruhe, Germany) for providing the

standardized extract EGb 761, Dr. Ikhlas Khan of the University of Mississippi for providing the

individual consitituents, and Dr. Sabine Heinhorst and Dr. Ken Curry of the University of

Southern Mississippi for reading the manuscript. This work was supported by a grant from

National Institutes of Health/National Center for Complementary and Alternative Medicine

(AT00293-01A2, YL), a grant from (Beaufour Ipsen, France) and The Innovation Award

from The University of Southern Mississippi (Y.L.).

REFERENCES

1. Kim, K. K., Kim, R., and Kim, S. H. (1998) Crystal structure of a small heat-shock protein.

Nature 394, 595�599

2. Valdez, M. M., Clark, J. I., Wu, G. J., and Muchowski, P. J. (2002) Functional similarities

between the small heat shock proteins Mycobacterium tuberculosis HSP 16.3 and human

alphaB-crystallin. Eur. J. Biochem. 269, 1806�1813

3. Link, C. D., Taft, A., Kapulkin, V., Duke, K., Kim, S., Fei, Q., Wood, D. E., and Sahagan,

B. G. (2003) Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer's

disease model. Neurobiol. Aging 24, 397�413

4. Yatin, S. M., Varadarajan, S., Link, C. D., and Butterfield, D. A. (1999) In vitro and in vivo

oxidative stress associated with Alzheimer�s amyloid beta-peptide (1-42). Neurobiol. Aging

20, 325�330; discussion 339�342

5. Leroux, M. R., Melki, R., Gordon, B., Batelier, G., and Candido, E. P. (1997) Structure-

function studies on small heat shock protein oligomeric assembly and interaction with

unfolded polypeptides. J. Biol. Chem. 272, 24646�24656

6. GuhaThakurta, D., Palomar, L., Stormo, G. D., Tedesco, P., Johnson, T. E., Walker, D. W.,

Lithgow, G., Kim, S., and Link, C. D. (2002) Identification of a novel cis-regulatory element

involved in the heat shock response in Caenorhabditis elegans using microarray gene

expression and computational methods. Genome Res. 12, 701�712

7. Link, C. D., Cypser, J. R., Johnson, C. J., and Johnson, T. E. (1999) Direct observation of

stress response in Caenorhabditis elegans using a reporter transgene. Cell Stress Chaperones

4, 235�242

8. DeFeudis, F. V. (1998) Ginkgo biloba extract (EGb 761): from chemistry to clinic. Publi

Ullstein Med. Weisbaden, Germany

9. Le Bars, P. L., Katz, M. M., Berman, N., Itil, T. M., Freedman, A. M., and Schatzberg, A. F.

(1997) A placebo-controlled, double-blind, randomized trial of an extract of Ginkgo biloba

for dementia. North American EGb Study Group. JAMA 278, 1327�1332

10. Le Bars, P. L., Kieser, M., and Itil, K. Z. (2000) A 26-week analysis of a double-blind,

placebo-controlled trial of the ginkgo biloba extract EGb 761 in dementia. Dement. Geriatr.

Cogn. Disord. 11, 230�237

11. Oken, B. S., Storzzbach, D. M., and Kaye, J. A. (1998) The efficacy of Ginkgo biloba on

cognitive function in Alzheiner disease. Arch. Neurol. 55, 1409�1415

12. Winter, J. C. (1998) The effects of an extract of Ginkgo biloba, EGb 761, on cognitive

behavior and longevity in the rat. Physiol. Behav. 63, 425�433

13. Christen, Y., Droy-Lefaix, M. T., Pasquier, C., and Packer, L. (2000) �Effects of Ginkgo

biloba extract (EGb761) on Alzheimer�s Disease.� In Free Radicals in Brain

Pathophysiology (pp. 411�425, Packer, L., ed.). Marcel Decker, New York

14. DeFeudis, F. V. (2002) Effects of Ginkgo biloba extract (EGb 761) on gene expression:

possible relevance to neurological disorders and age-associated cognitive impairment. Drug

Dev. Res. 57, 214�235

15. Luo, Y. (2001) Ginkgo biloba neuroprotection: Therapeutic implications in Alzheimer's

disease. J. Alzheimers Dis. 3, 401�407

16. Watanabe, C. M., Wolffram, S., Ader, P., Rimbach, G., Packer, L., Maguire, J. J., Schultz,

P. G., and Gohil, K. (2001) The in vivo neuromodulatory effects of the herbal medicine

ginkgo biloba. Proc. Natl. Acad. Sci. USA 98, 6577�6580

17. Curtis-Prior, P., Vere, D., and Fray, P. (1999) Therapeutic value of Ginkgo biloba in

reducing symptoms of decline in mental function. J. Pharm. Pharmacol. 51, 535�541

18. Mix, J. A., and Crews, W. D., Jr. (2002) A double-blind, placebo-controlled, randomized

trial of Ginkgo biloba extract EGb 761 in a sample of cognitively intact older adults:

neuropsychological findings. Hum. Psychopharmacol. 17, 267�277

19. Solomon, P. R., Adams, F., Silver, A., Zimmer, J., and DeVeaux, R. (2002) Ginkgo for

memory enhancement: a randomized controlled trial. JAMA 288, 835�840

20. Luo, Y., Smith, J. V., Paramasivam, V., Burdick, A., Curry, K. J., Buford, J. P., Khan, I.,

Netzer, W. J., Xu, H., and Butko, P. (2002) Inhibition of amyloid-beta aggregation and

caspase-3 activation by the Ginkgo biloba extract EGb761. Proc. Natl. Acad. Sci. USA 99,

12197�12202

21. Smith, J. V., Burdick, A. J., Golik, P., Khan, I., Wallace, D., and Luo, Y. (2002) Anti-

apoptotic properties of Ginkgo biloba extract EGb 761 in differentiated PC12 cells. Cell Mol

Biol 48, 699�707

22. Wu, Z., Smith, J. V., Paramasivam, V., Butko, P., Khan, I., Cypser, J. R., and Luo, Y.

(2002) Ginkgo biloba extract EGb 761 increases stress resistance and extends life span of

caenoraibditis elegans. Cell Mol Biol (Noisy-le-grand) 48, 725�731

23. Ward, C. P., Redd, K., Williams, B. M., Caler, J. R., Luo, Y., and McCoy, J. G. (2002)

Ginkgo biloba extract. Cognitive enhancer or antistress buffer. Pharmacol. Biochem. Behav.

72, 913�922

24. Ganzera, M., Zhao, J., and Khan, I. A. (2001) Analysis of terpenelactones in Ginkgo biloba

by high performance liquid chromatography and evaporative light scattering detection.

Chem. Pharm. Bull. (Tokyo) 49, 1170�1173

25. Royall, J. A., and Ischiropoulos, H. (1993) Evaluation of 2',7'-dichlorofluorescin and

dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial

cells. Arch. Biochem. Biophys. 302, 348�355

26. Harman, D. (1957) Ageing: a theory based on free radical and radiation chemistry. J.

Gerontol. 2, 298�300

27. Butterfield, D. A., Howard, B., Yatin, S., Koppal, T., Drake, J., Hensley, K., Aksenov, M.,

Aksenova, M., Subramaniam, R., Varadarajan, S., et al. (1999) Elevated oxidative stress in

models of normal brain aging and Alzheimer's disease. Life Sci. 65, 1883�1892

28. Smith, J. V., and Luo, Y. (2003) Elevation of oxidative free radicals in Alzheimer's Disease

models can be attenuated by Ginkgo biloba extract EGb 761. J. Alzheimer�s Disease, 4 (in

press)

29. Larsen, P. L. (1993) Aging and resistance to oxidative damage in Caenorhabditis elegans.

Proc. Natl. Acad. Sci. USA 90, 8905�8909

30. Walker, G. A., White, T. M., McColl, G., Jenkins, N. L., Babich, S., Candido, E. P.,

Johnson, T. E., and Lithgow, G. J. (2001) Heat shock protein accumulation is upregulated in

a long-lived mutant of Caenorhabditis elegans. J. Gerontol. A Biol. Sci. Med. Sci. 56, B281�

B287

31. Rogalla, T., Ehrnsperger, M., Preville, X., Kotlyarov, A., Lutsch, G., Ducasse, C., Paul, C.,

Wieske, M., Arrigo, A. P., Buchner, J., et al. (1999) Regulation of Hsp27 oligomerization,

chaperone function, and protective activity against oxidative stress/tumor necrosis factor

alpha by phosphorylation. J. Biol. Chem. 274, 18947�18956

32. Horwitz, J. (1992) Alpha-crystallin can function as a molecular chaperone. Proc. Natl. Acad.

Sci. USA 89, 10449�10453

33. Mehlen, P., Schulze-Osthoff, K., and Arrigo, A. P. (1996) Small stress proteins as novel

regulators of apoptosis. Heat shock protein 27 blocks Fas/APO-1- and staurosporine-induced

cell death. J. Biol. Chem. 271, 16510�16514

34. Mehlen, P., Mehlen, A., Godet, J., and Arrigo, A. P. (1997) hsp27 as a switch between

differentiation and apoptosis in murine embryonic stem cells. J. Biol. Chem. 272, 31657�

31665

35. Taricani, L., Feilotter, H. E., Weaver, C., and Young, P. G. (2001) Expression of hsp16 in

response to nucleotide depletion is regulated via the spc1 MAPK pathway in

Schizosaccharomyces pombe. Nucleic Acids Res. 29, 3030�3040

36. Christen, Y., and Maixent, J. M. (2002) What is Ginkgo biloba extract EGb 761? An

overview�from molecular biology to clinical medicine. Cell Mol Biol 48, 601�611

37. Schutte, A., Topp, S. A., Knoefel, W. T., Brilloff, S., Mueller, L., Rogiers, X., and

Gundlach, M. (2001) Influence of Ginkgo Biloba extract (EGB 761) on expression of EGR-

1 mRNA and HSP-70 mRNA after warm ischemia in the rat liver. Transplant. Proc. 33,

3724�3725

38. Soulie, C., Nicole, A., Christen, Y., and Ceballos-Picot, I. (2002) The Ginkgo biloba extract

EGb 761 increases viability of hnt human neurons in culture and affectsthe expression of

genes implicated in the stress response. Cell Mol Biol 48, 641�646

39. Westman, J., Drieu, K., and Sharma, H. S. (2000) Antioxidant compounds EGB-761 and

BN-520 21 attenuate heat shock protein (HSP 72 kD) response, edema and cell changes

following hyperthermic brain injury. An experimental study using immunohistochemistry in

the rat. Amino Acids 19, 339�350

40. Freshney, N. W., Rawlinson, L., Guesdon, F., Jones, E., Cowley, S., Hsuan, J., and

Saklatvala, J. (1994) Interleukin-1 activates a novel protein kinase cascade that results in the

phosphorylation of Hsp27. Cell 78, 1039�1049

41. Guesdon, F., Freshney, N., Waller, R. J., Rawlinson, L., and Saklatvala, J. (1993)

Interleukin 1 and tumor necrosis factor stimulate two novel protein kinases that

phosphorylate the heat shock protein hsp27 and beta-casein. J. Biol. Chem. 268, 4236�4243

42. Kato, K., Ito, H., Kamei, K., Iwamoto, I., and Inaguma, Y. (2002) Innervation-dependent

phosphorylation and accumulation of alphaB- crystallin and Hsp27 as insoluble complexes

in disused muscle. FASEB J. 16, 1432�1434

43. Wadsworth, T. L., McDonald, T. L., and Koop, D. R. (2001) Effects of Ginkgo biloba

extract (EGb 761) and quercetin on lipopolysaccharide-induced signaling pathways involved

in the release of tumor necrosis factor-alpha. Biochem. Pharmacol. 62, 963�974

44. Butterfield, D. A., Howard, B. J., Yatin, S., Allen, K. L., and Carney, J. M. (1997) Free

radical oxidation of brain proteins in accelerated senescence and its modulation by N-tert-

butyl-alpha-phenylnitrone. Proc. Natl. Acad. Sci. USA 94, 674�678

45. Hassan, H. M., and Fridovich, I. (1979) Intracellular production of superoxide radical and of

hydrogen peroxide by redox active compounds. Arch. Biochem. Biophys. 196, 385�395

46. Chao, S. H., Greenleaf, A. L., and Price, D. H. (2001) Juglone, an inhibitor of the peptidyl-

prolyl isomerase Pin1, also directly blocks transcription. Nucleic Acids Res. 29, 767�773

Received June 11, 2003; accepted August 5, 2003.

Fig. 1

Figure 1. A) Representative epifluorescence image of wild-type and transgenic Caenorhabditis elegans CL2070 (hsp-16-

2/GFP) under heat-shock treatment (HS). Both the wild-type (a) and the transgenic C. elegans (b) strains grown on NGM

at 20oC in a temperature-controlled incubator were exposed to thermal stress (35oC for 2 h). The photographs include the

entire anterior part of the pharynx showing both nerve rings. B, C) EGb 761 modulates hsp16-2 expression induced by

heat (HS) (B) or juglone (Jug) (C). B) CL2070 (hsp-16-2/GFP) worms, grown at 20oC, were treated with (b) or without

(a) 100 µg/ml EGb 761 for 48 h starting at 2 days of age. The worms were exposed to 35oC for 2 h and transferred to 20oC

for 4 h to recover before fluorescence microscopy. C) CL2070 worms fed with (b) or without (a) 100 µg/ml EGb 761 for

48 h followed by 160 µM juglone challenge for 24 h. D) Control worms (Ctrl, CL1234) expressing constitutive GFP

protein were fed with or without EGb 761 for 48 h. All of the inset images display endogenous GFP fluorescence, either

induced (B, C) or constituently expressed (D). For quantifying a population of GFP reporter animals, each 40× image was

analyzed using ImageProPlus software. Data are expressed as GFP mean pixel density obtained from at least four

independent experiments with at least 24 worms in each experimental group. *Statistically significant (independent t test,

P<0.05); ***statistically significant, P<0.0001.

Fig. 2

Figure 2. Effect of EGb 761 on stress response in CL2070 hsp-16-2/GFP Caenorhabditis elegans. A) Survival assay of hsp-16-2/GFP C. elegans under thermal stress. Four-day-old worms, raised at 20°C either untreated (open circles) or treated with 100 µg/ml EGb 761 (filled circles), were transferred to 35°C, and survival was measured at regular intervals every hour. B) Survival assay of hsp-16-2/GFP C. elegans under an acute, lethal dose of oxidative stress. The CL2070 worms were untreated (open squares) or pretreated (filled squares) with EGb 761 for 48 h and transferred onto the medium containing 160 µM juglone, and survival was scored at regular intervals (every 30 min). Standard errors for each data point were calculated using Origin software. Total number of worms in the experimental groups was 189 (control) and 179 (EGb 761).

Fig. 3

Figure 3. Attenuation of hsp-16-2 expression by post-juglone administration of EGb 761 or by other known antioxidants. A) The hsp-16-2/GFP worms treated with 100 µg/ml EGb 761 for 48 h either simultaneously (Co-jug+EGb) or immediately after (Post-Jug+EGb) exposed to 160 µM of juglone for 24 h were examined with fluorescence microscopy. B) The hsp-16-2/GFP worms treated with known antioxidants vitamin C (L-ascorbic acid) or flavonoid fractions of EGb 761 (FLV) for 48 h were analyzed by examination of GFP reporter transgene expression. Data are expressed as GFP green fluorescent mean pixel density. Data of each graph are from three independent experiments of at least 10 worms in each group in each experiment. *Statistically significant (unpaired t test, P<0.05).

Fig. 4

Figure 4. Effect of EGb 761 on intracellular H2O2-related reactive oxygen species (ROS) basal levels in hsp-16-2/GFP transgenic Caenorhabditis elegans. Age-synchronized groups of transgenic C. elegans, maintained and collected as described in Materials and Methods, were assayed at day 4 of age after treated with or without (Ctrl) 100 µg/ml of EGb 761 (EGb) or flavonoids (FLV) or L-ascorbic acid (VitC) for 48 h. The worms were then analyzed for the levels of H2O2-related ROS by incubating with 50 µM DCF-DA for 2.5 h, followed by measurement of fluorescent DCF production. Results are expressed as DCF fluorescence relative to untreated controls. *Statistically significant (independent t test, P<0.05). Results are obtained from three independent experiments with a total of 300 worms.

Fig. 5

Figure 5. Schematic diagram of postulated anti-stress mechanism of EGb 761 in Caenorhabditis elegans. Heat shock and oxidative stressors induce expression of hsp-16-2 as a result of increased levels of reactive oxidative species (ROS) and protein damages, which lead to increased death of C. elegans. Our results show that EGb 761 suppresses hsp-16-2 expression as an indication that EGb 761 decreases cellular stress resulting from exogenous stressors, therefore increasing stress resistance and survival. However, how EGb 761 modulates the expression of hsp-16-2 and the physiological function of hsp-16-2 still remains to be determined.